Cholecystokinin antagonists

ABSTRACT

Benzodiazepine analogs of the formula: ##STR1## are disclosed which are antagonists of gastrin and cholecystokinin (CCK).

CROSS REFERENCE

This is a continuation-in-part application of U.S. Ser. No. 07/812,876filed on Dec. 20, 1991, now abandoned, which is a continuationapplication of U.S. Ser. No. 07/683,407 filed on Apr. 10, 1991, nowabandoned.

FIELD OF THE INVENTION

This invention relates to the discovery of Benzodiazepine analogs ofFormula I for use as antagonists of cholecystokinin (CCK) and gastrinwhen administered to animals, preferably humans.

BACKGROUND OF THE INVENTION

The Benzodiazepine analogs of Formula I of this invention are useful intreating various diseases caused by an excess of CCK or gastrin.Cholecystokinins (CCK) and gastrin are structurally relatedneuropeptides which exist in gastrointestinal tissue and in the centralnervous system (see, V. Mutt, Gastrointestinal Hormones, G. B. J. Glass,Ed., Raven Press, New York, p. 169 and G. Nission, ibid, p. 127.

Cholecystokinins include CCK-33, a neuropeptide of thirty-three aminoacids in its originally isolated form (see, Mutt and Jorpes, Biochem. J.125, 678 (1971)), its carboxyl terminal octapeptide, CCK-8 (also anaturally-occurring neuropeptide and the minimum fully active sequence),and 39- and 12-amino acid forms. Gastrin occurs in 34-, 17- and 14-aminoacid forms, with the minimum active sequence being the C-terminaltetrapeptide, Trp-Met-Asp-Phe-NH₂, which is the common structuralelement shared by both CCK and gastrin.

CCK's are believed to be physiological satiety hormones, therebypossibly playing an important role in appetite regulation (G. P. Smith,Eating and Its Disorders, A. J. Stunkard and E. Stellar, Eds, RavenPress, New York, 1984, p. 67), as well as also stimulating colonicmotility, gall bladder contraction, pancreatic enzyme secretion, andinhibiting gastric emptying. They reportedly co-exist with dopamine incertain mid-brain neurons and thus may also play a role in thefunctioning of dopaminergic systems in the brain, in addition to servingas neurotransmitters in their own right (see: A. J. Prange et al.,"Peptides in the Central Nervous System", Ann. Repts. Med. Chem. 17, 31,33 [1982] and references cited therein; J. A. Williams, Biomed. Res. 3107 [1982]; and J. E. Morley, Life Sci. 30, 479, [1982]).

The primary role of gastrin, on the other hand, appears to bestimulation of the secretion of water and electrolytes in the stomach,and, as such, it is involved in control of gastric acid and pepsinsecretion. Other physiological effects of gastrin then include increasedmucosal blood flow and increased antral motility. Rat studies have shownthat gastrin has a positive trophic effect on the gastric mucosa, asevidenced by increased DNA, RNA and protein synthesis.

Antagonists to CCK and to gastrin have been useful for preventing andtreating CCK-related and/or gastrin-related disorders of thegastrointestinal (GI) and central nervous (CNS) systems of animals,preferably mammals, and especially those of humans. Just as there issome overlap in the biological activities of CCK and gastrin,antagonists also tend to have affinity for both receptors. In apractical sense, however, there is enough selectivity for the differentreceptors that greater activity against specific CCK- or gastrin-relateddisorders can often also be identified.

Selective CCK antagonists are themselves useful in treating CCK-relateddisorders of the appetite regulatory systems of animals as well as inpotentiating and prolonging opiate-mediated analgesia, thus havingutility in the treatment of pain [see P. L. Faris et al., Science 226,1215 (1984)]. Selective gastrin antagonists are useful in the modulationof CNS behavior, as a palliative for gastrointestinal neoplasms, and inthe treatment and prevention of gastrin-related disorders of thegastrointestinal system in humans and animals, such as peptic ulcers,Zollinger-Ellison syndrome, antral G cell hyperplasia and otherconditions in which reduced gastrin activity is of therapeutic value.See e.g. U.S. Pat. No. 4,820,834. It is further expected that the CCKantagonists of Formula I are useful anxiolytic agents particularly inthe treatment of panic and anxiety disorders.

Since CCK and gastrin also have trophic effects on certain tumors [K.Okyama, Hokkaido J. Med. Sci., 60, 206-216 (1985)], antagonists of CCKand gastrin are useful in treating these tumors [see, R. D. Beauchamp etal., Ann. Surg., 202, 303 (1985)].

Distinct chemical classes of CCK-receptor antagonists have been reported[R. Freidinger, Med. Res. Rev. 9, 271 (1989)]. The first class comprisesderivatives of cyclic nucleotides, of which dibutyryl cyclic GMP hasbeen shown to be the most potent by detailed structure-function studies(see, N. Barlas et al., Am. J. Physiol., 242, G 161 (1982) and P.Robberecht et al., Mol., Pharmacol., 17, 268 (1980)).

The second class comprises peptide antagonists which are C-terminalfragments and analogs of CCK, of which both shorter(Boc-Met-Asp-Phe-NH₂, Met-Asp-Phe-NH₂), and longer (Cbz-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-NH₂) C-terminal fragments of CCK can function asCCK antagonists, according to recent structure-function studies (see, R.T. Jensen et al., Biochem. Biophys. Acta., 757, 250 (1983), and M.Spanarkel et al., J. Biol. Chem., 258, 6746 (1983)). The latter compoundwas recently reported to be a partial agonist [see, J. M. Howard et al.,Gastroenterology 86(5) Part 2, 1118 (1984)].

The third class of CCK-receptor antagonists comprises the amino acidderivatives: proglumide, a derivative of glutaramic acid, and the N-acyltryptophans including para-chlorobenzoyl-L-tryptophan (benzotript),[see, W. F. Hahne et al., Proc. Natl. Acad. Sci. U.S.A., 78, 6304(1981), R. T. Jensen et al., Biochem. Biophys. Acta., 761, 269 (1983)].All of these compounds, however, are relatively weak antagonists of CCK(IC₅₀ : generally 10⁻⁴ M[although more potent analogs of proglumide havebeen recently reported in F. Makovec et al., Arzneim-Forsch Drug Res.,35 (II), 1048 (1985) and in German Patent Application DE 3522506A1], butdown to 10⁻⁶ M in the case of peptides), and the peptide CCK-antagonistshave substantial stability and absorption problems.

In addition, a fourth class consists of improved CCK-antagonistscomprising a nonpeptide of novel structure from fermentation sources [R.S. L. Chang et al., Science, 230, 177-179 (1985)] and 3-substitutedbenzodiazepines based on this structure [published European PatentApplications 167 919, 167 920 and 169 392, B. E. Evans et al, Proc.Natl. Acad. Sci. U.S.A., 83, p. 4918-4922 (1986) and R. S. L. Chang etal, ibid, p. 4923-4926] have also been reported.

No really effective receptor antagonists of the in vivo effects ofgastrin have been reported (J. S. Morley, Gut Pept. Ulcer Proc.,Hiroshima Symp. 2nd, 1983, p. 1), and very weak in vitro antagonists,such as proglumide and certain peptides have been described [(J.Martinez, J. Med. Chem. 27, 1597 (1984)]. Recently, however,pseudopeptide analogs of tetragastrin have been reported to be moreeffective gastrin antagonists than previous agents [J. Martinez et al.,J. Med. Chem., 28, 1874-1879 (1985)].

A new class of Benzodiazepine antagonist compounds has further beenreported which binds selectively to brain CCK (CCK-B) and gastrinreceptors [see M. Bock et al., J. Med. Chem., 32, 13-16 (1989)]. Onecompound of interest reported in this reference to be a potent andselective antagonist of CCK-B receptors is(R)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N.sup.1-(3-methylphenyl) urea (See U.S. Pat. No. 4,820,834.) One disadvantageof the new CCK-B compound reported in Bock et al., J. Med. Chem., 32,13-16 (1989) and U.S. Pat. No. 4,820,834, is that these CCK-B compoundsare poorly water soluble.

It is, therefore, an object of the present invention to provideantagonists of CCK and gastrin. If an antagonist compound could beprepared which would bind with the cell surface receptor of CCK orgastrin, then the antagonist compounds of this invention could be usedto block the effect of CCK and gastrin. Another object of the presentinvention is to provide novel CCK and gastrin antagonist compounds whichare water soluble. Other objects of the present invention are to providemethods of inhibiting the action of CCK and gastrin through theadministration of novel benzodiazepine analog compounds. The above andother object are accomplished by the present invention in the mannermore fully described below.

SUMMARY OF THE INVENTION

The present invention provides Benzodiazepine analogs of the formula:##STR2## for use as antagonists of CCK and gastrin. The above-mentionedcompounds can be used in a method of acting upon a CCK and/or gastrinreceptor which comprises administering a therapeutically effective butnon-toxic amount of such compound to an animal, preferably a human. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and, dispersed therein, an effective but non-toxic amount ofsuch compound is another aspect of this invention.

DETAILED DESCRIPTION OF THE INVENTION

Benzodiazepine analogs of Formula I provide antagonists of CCK andgastrin. The present invention further provides novel CCK and gastrinantagonist compound which are water soluble. The Benzodiazepine analogsof Formula I are useful in a method of antagonizing the binding of CCKto CCK receptors or antagonizing the binding of gastrin to gastrinreceptors. The novel Benzodiazepine analogs of the present invention areillustrated by compounds having the formula: ##STR3## wherein: R¹ is H,--(CH₂)₂ --CO₂ CH₃, or

--(CH₂)₂ --CO₂ H;

R² is ##STR4## R³ is absent, one or two of Halogen or CH₃ ; R⁴ isabsent, one or two of Halogen or CH₃ ;

R⁵ is ##STR5## or the optical isomers, prodrugs or pharmaceuticallyacceptable salts thereof.

Preferred compounds of this invention as set forth in the Examples are:

N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-carboxyethyl-N'-{[3-methylphenyl]-urea};and

N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-carboxyethyl-N'-{[3-methylphenyl]-urea}.

It will be appreciated that formula (I) is intended to embrace allpossible isomers, including optical isomers, and mixtures thereof,including racemates.

The present invention includes within its scope prodrugs of thecompounds of formula I above. In general, such prodrugs will befunctional derivatives of the compounds of formula I which are readilyconvertible in vivo into the required compound of formula I.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in "Design of Prodrugs",ed. H. Bungaard, Elsevier, 1985.

The pharmaceutically acceptable salts of the compounds of Formula Iinclude the conventional non-toxic salts or the quarternary ammoniumsalts of the compounds of Formula I formed, e.g., from non-toxicinorganic or organic acids. For example, such conventional non-toxicsalts include those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the compounds of Formula I which contain a basic oracidic moiety by conventional chemical methods. Generally, the salts areprepared by reacting the free base or acid with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidor base in a suitable solvent or various combinations of solvents.

The pharmaceutically acceptable salts of the acids of Formula I are alsoreadily prepared by conventional procedures such as treating an acid ofFormula I with an appropriate amount of a base, such as an alkali oralkaline earth metal hydroxide e.g. sodium, potassium, lithium, calcium,or magnesium, or an organic base such as an amine, e.g.,dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine,benzylamine and the like, or a quaternary ammonium hydroxide such astetramethylammonium hydroxide and the like.

The compounds of Formula I antagonize CCK and/or gastrin and are usefulas pharmaceutical agents for animals, preferably for mammals, and mostespecially for humans, for the treatment and prevention ofgastrointestinal disorders and central nervous system disorders.

Examples of such gastrointestinal disorders include ulcers, such aspeptic and gastrointestinal ulcers, irritable bowel syndrome,gastroesophagenal reflux disease or excess pancreatic or gastrinsecretion, acute pancreatitis, or motility disorders, Zollinger-Ellisonsyndrome, and antral and cell hyperplasia.

Examples of central nervous system disorders include central nervoussystem disorders caused by CCK interaction with dopamine, such asneuroleptic induced tardive dyskinesia, Parkinson's disease,schizophrenia, other psychosis or Gilles de la Tourette syndrome, anddisorders of appetite regulatory systems.

The compounds of Formula I may further be useful in the treatment orprevention of additional central nervous system disorders includingneurological and psychiatric disorders. Examples of such central nervoussystem disorders include anxiety disorders and panic disorders, whereinCCK and/or gastrin is involved. Additional examples of central nervoussystem disorders include panic syndrome, anticipatory anxiety, phobicanxiety, panic anxiety, chronic anxiety, and endogenous anxiety.

The compounds of Formula I may further be useful in the treatment ofoncologic disorders wherein CCK or gastrin may be involved. Examples ofsuch oncologic disorders include small cell adenocarcinomas and primarytumors of the central nervous system glial and neuronal cells. Examplesof such adenocarcinomas and tumors include, but are not limited to,tumors of the lower esophagus, stomach, intestine, colon and lung,including small cell lung carcinoma.

The compounds of Formula I may further be used to control pupilconstriction in the eye. The compounds may be used for therapeuticpurposes during eye examinations and intraocular surgery in order toprevent miosis. The compounds may further be used to inhibit miosisoccurring in association with iritis, uveitis and trauma.

The compounds of Formula I are also useful for directly inducinganalgesia, opiate or non-opiate mediated, as well as anesthesia or lossof the sensation of pain.

The compounds of Formula I may further be useful for preventing ortreating the withdrawal response produced by chronic treatment or abuseof drugs or alcohol. Such drugs include, but are not limited to cocaine,alcohol or nicotine.

The compounds of formula (I) may also be useful as neuroprotectiveagents, for example, in the treatment and/or prevention ofneurodegenerative disorders arising as a consequence of suchpathological conditions as stroke, hypoglycaemia, cerebral palsy,transient cerebral ischaemic attack, cerebral ischaemia during cardiacpulmonary surgery or cardiac arrest, perinatal asphyxia, epilepsy,Huntington's chorea, Alzheimer's disease, Amyotrophic LaterialSclerosis, Parkinson's disease, Olivo-pontocerebellar atrophy, anoxiasuch as from drowing, spinal cord and head injury, and poisoning byneurotoxins, including environmental neurotoxins.

The present invention also encompasses a pharmaceutical compositionuseful in the treatment of CCK and/or gastrin disorders comprising theadministration of a therapeutically effective but non-toxic amount ofthe compounds of Formula I, with or without pharmaceutically acceptablecarriers or diluents.

The compounds of Formula I, may be administered to animals, preferablyto mammals, and most especially to a human subject either alone or,preferably, in combination with pharmaceutically-acceptable carriers ordiluents, optionally with known adjuvants, such as alum, in apharmaceutical composition, according to standard pharmaceuticalpractice. The compounds can be administered orally or parenterally,including intravenous, intramuscular, intraperitoneal, subcutaneous andtopical administration.

For oral use of an antagonist of CCK, according to this invention, theselected compounds may be administered, for example, in the form oftablets or capsules, or as an aqueous solution or suspension. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch, and lubricating agents, such as magnesiumstearate, are commonly added. For oral administration in capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring agents may be added. For intramuscular,intraperitoneal, subcutaneous and intravenous use, sterile solutions ofthe active ingredient are usually prepared, and the pH of the solutionsshould be suitably adjusted and buffered. For intravenous use, the totalconcentration of solutes should be controlled in order to render thepreparation isotonic.

When a compound according to Formula I is used as an antagonist of CCKor gastrin in a human subject, the daily dosage will normally bedetermined by the prescribing physician with the dosage generallyvarying according to the age, weight, and response of the individualpatient, as well as the severity of the patient's symptoms. However, inmost instances, an effective daily dosage will be in the range of fromabout 0.005 mg/kg to about 50 mg/kg of body weight, and preferably, offrom about 0.05 mg/kg to about 50 mg/kg of body weight, and mostpreferably, of from about 0.5 mg/kg to about 20 mg/kg of body weightadministered in single or divided doses.

In some cases, however, it may be necessary to use dosage levels outsidethese limits. For example, doses as low as about 1 ng/kg, about 0.005 μgto about 0.05 μg, or about 100 ng to about 100 μg/kg may beadministered.

In the effective treatment of panic syndrome, panic disorder, anxietydisorder and the like, preferably about 0.05 mg/kg to about 1.0 mg/kg ofCCK antagonist maybe administered orally (p.o.), administered in singleor divided doses per day (b.i.d.). Other routes of administration arealso suitable.

For directly inducing analgesia, anesthesia or loss of pain sensation,the effective dosage range is preferably from about 100 ng/kg to about 1mg/kg by intraperitoneal administration. Oral administration is analternative route, as well as others.

In the treatment of irritable bowel syndrome, preferably about 0.1 to 10mg/kg of CCK antagonist is administered orally (p.o.), administered insingle or divided doses per day (b.i.d.). Other routes of administrationare also suitable.

The use of a gastrin antagonist as a tumor palliative forgastrointestinal neoplasma with gastrin receptors, as a modulator ofcentral nervous activity, treatment of Zollinger-Ellison syndrome, or inthe treatment of peptic ulcer disease, an effective dosage is preferablyfrom about 0.1 to about 10 mg/kg administered one-to-four times daily isindicated.

Because these compounds antagonize the function of CCK in animals, theymay also be used as feed additives to increase the food intake ofanimals in daily dosage preferably from about 0.05 mg/kg to about 50mg/kg of body weight.

The compounds of Formula I may be prepared according to the reactionschemes as set forth below. ##STR6##

1. CCK Receptor Binding (Pancreas)

CCK-8 sulphated was radiolabelled with ¹²⁵ I-Bolton Hunter reagent (2000Ci/mmole). Receptor binding was performed according to Chang and Lotti(Proc. Natl. Acad. Sci. 83, 4923-4926, 1986) with minor modifications.

Male Sprague-Dawley rats (150-200 g) were sacrificed by decapitation.The whole pancreas was dissected free of fat tissue and was homogenizedin 25 volumes of ice-cold 10 mM Hepes buffer with 0.1% soya bean trypsininhibitor (pH 7.4 at 25° C.) with a Kinematica Polytron. The homogenateswere centrifuged at 47,800 g for 10 min. Pellets were resuspended in 10volumes of binding assay buffer (20 mM Hepes, 1 mM EGTA, 5 mM MgCl₂, 150mM NaCl, bacitracin 0.25 mg/ml, soya bean trypsin inhibitor 0.1 mg/ml,and bovine serum albumin 2 mg/ml, pH 6.5 at 25° C.) using a teflonhomogenizer, 15 strokes at 500 rpm. The homogenate was further dilutedin binding assay buffer to give a final concentration of 0.5 mg originalwet weight/1 ml buffer. For the binding assay, 50 μl of buffer (fortotal binding) or unlabeled CCK-8 sulfated to give a final concentrationof 1 μM (for nonspecific binding) or the compounds of Formula I (fordetermination of inhibition of ¹²⁵ I-CCK binding) and 50 μl of 500 pM¹²⁵ I-CCK-8 (i.e. 50 pM final concentration) were added to 400 μl of themembrane suspensions in microfuge tubes. All assays were run induplicate. The reaction mixtures were incubated at 25° C. for 2 hoursand the reaction terminated by rapid filtration (Brandell 24 well cellharvester) over Whatman GF/C filters, washing 3×4 mls with ice-cold 100mM NaCl. The radioactivity on the filters was counted with a LKB gammacounter.

2. CCK Receptor Binding (Brain)

CCK-8 sulphated was radiolabelled and the binding was performedaccording to the description for the pancreas method with minormodifications.

Male Hartley guinea pigs (300-500 g) were sacrificed by decapitation andthe cortex was removed and homogenized in 25 mL ice-cold 0.32M sucrose.The homogenates were centrifuged at 1000 g for 10 minutes and theresulting supernatant was recentrifuged at 20,000 g for 20 minutes. TheP₂ pellet was resuspended in binding assay buffer (20 mMN-2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid (HEPES), 5 mMMgCl₂, 0.25 mg/ml bacitracin, 1 mM ethyleneglycol-bis-(β-aminoethylether-N,N'-tetraacetic acid) (EGTA)pH 6.5 at 25°C., using a teflon homogenizer (5 strokes at 500 rpm) to give a finalconcentration of 10 mg original wet weight 11.2 mls buffer. For thebinding assay, 50 μl of buffer (for total binding) or unlabeled CCK-8sulfate to give a final concentration of 1 μM (for nonspecific binding)or the compounds of Formula I (for determination of inhibition of ¹²⁵I-CCK-8 binding) and 50 μl of 500 pM ¹²⁵ I-CCK-8 (i.e. finalconcentration of 50 pM) were added to 400 μl of the membrane suspensionsin microfuge tubes. All assays were run in duplicate. The reactionmixtures were incubated at 25° C. for 2 hours and then the reaction wasterminated on Whatman GF/C filters by rapid filtration (Brandell 24 wellcell Harvester) with 3×5 ml washes of cold 100 mM NaCl. Theradioactivity on the filters was then counted with a LKB gamma counter.

5. Gastrin Antagonism

Gastrin antagonist activity of compounds of Formula I is determinedusing the following assay.

A. Gastrin Receptor Binding in Guinea Pig Gastric Glands

Preparation of guinea pig gastric mucosal glands

Guinea pig gastric muscosal glands were prepared by the procedure ofChang et al., Science 230, 177-179 (1985) with slight modification.Gastric mucosa from guinea pigs (300-500 g body weight, male Hartley)were isolated by scraping with a glass slide after washing stomach inice-cold, aerated buffer consisting of the following: 130 mM NaCl, 12 mMNaHCO₃, 3 mM NaH₂ PO₄, 3 mM Na₂ HPO₄, 3 mM K₂ HPO₄, 2 mM MgSO₄, 1 mMCaCl₂, 5 mM glucose and 4 mM L-glutamine, 50 mM HEPES, 0.25 mg/mlbacitracin, 0.10 mg/ml soya bean trypsin inhibitor, 0.1 mg/ml bovineserum albumin, at pH 6.5, and then incubated in a 37° C. shaking waterbath for 40 minutes in buffer containing 1 mg/ml collagenase and bubbledwith 95% O₂ and 5% CO₂. The tissues were passed twice through a 5 mlsyringe to liberate the gastric glands, and then filtered through Nitex#202 gauge nylon mesh. The filtered glands were centrifuged at 272 g for5 minutes and washed twice by resuspension in 25 ml buffer andcentrifugation.

B. Binding Studies

The washed guinea pig gastric glands prepared as above were resuspendedin 25 ml of standard buffer. For binding studies, to 250 μl of gastricglands, 30 μl of buffer (for total binding) or gastrin (3 μM finalconcentration, for nonspecific binding) or test compound and 20 μl of¹²⁵ I-gastrin (NEN, 2200 Ci/mmole, 0.1 nM final concentration) wereadded. AV assays were run in triplicate. The tubes were aerated with 95%O₂ and 5% CO₂ and capped. The reaction mixtures after incubation at 25°C. for 30 minutes in a shaking water bath were rapidly filtered(Brandell 24 well cell harvester) over Whatman and G/F B filterspresoaked in assay buffer and immediately washed further with 3×4 ml of100 mM ice cold NaCl. The radioactivity on the filters was measuredusing a LKB gamma counter.

In Vitro Results

Effect of The Compounds of Formula I on ¹²⁵ I-CCK-8 receptor binding

The preferred compounds of Formula I are those which produceddose-dependent inhibition of specific ¹²⁵ I-CCK-8 binding as defined asthe difference between total and non-specific (i.e. in the presence of 1μm CCK) binding.

Drug displacement studies were performed with at least 10 concentrationsof compounds of formula 1 and the IC₅₀ values were determined byregression analysis. IC₅₀ refers to the concentration of the compoundrequired to inhibit 50% of specific binding of ¹²⁵ I-CCK-8.

The data in Table I were obtained for compounds of Formula I.

                  TABLE I                                                         ______________________________________                                        CCK RECEPTOR BINDING RESULTS                                                  IC.sub.50 (μM)                                                             Compound  .sup.125 I-CCK                                                                           .sup.25 I-CCK                                                                          .sup.25 I-Gastrin                               of Ex #   Pancreas   Brain    Gastric Glands                                  ______________________________________                                        1         >3         3.68     N.D.                                            2         >3         0.51     N.D.                                            ______________________________________                                         N.D. = NO DATA                                                           

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof.

EXAMPLE 1 Synthesis ofN-{1,3-Dihyro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-carboxyethyl-N'-{[3-methylphenyl]-urea}A.N-{1,3-Dihyro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-methylcarboxyethyl-N'-{[3-methylphenyl]-urea}

To a solution of1,3-dihyro-3(R,S)-amino-5-phenyl-2H-1,4-benzodiazepin-2-one (100 mg,0.377 mmole) in 2 ml of toluene was added 56 mg (0.189 mmole)triphosgene and 56 μL of triethylamine (0.377 mmole). After 10 minutes,100 mg (0.517 mmole) of N-methoxycarbonylethyl-m-toluidine andtriethylamine (56 μL, 0.377 mmole) were added and stirring was continuedfor 2 additional hours. The reaction mixture was concentrated todryness. Ethyl acetate and water were added to the residue. The layerswere separated and the aqueous layer was extracted with ethyl acetate.The combined organic extracts were dried (sodium sulfate) andconcentrated to give approximately 150 mg of crude product. Preparativethick layer chromatography on 0.5 mm×20 cm×20 cm precoated silica gelplates (ethyl acetatehexane, 1:1 v/v elution) afforded 60 mg of theanalytical product after it was crystallized from petroleum ether: m.p.197°-198° C. HPLC=99.3% pure at 214 nm; TLC R_(f) =0.30 (EtOAc-hexane,1:1).

NMR (DMSO-D₆): Consistent with structure assignment and confirmspresence of solvent.

FAB MS: 485 (M⁺ +1).

Analysis for C₂₈ H₂₈ N₄ O₄ : Calculated: C, 69.40, H, 5.82, N, 11.56.Found: C, 69.19, H, 5.96, N, 11.52.

B.N-}1,3-Dihyro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-carboxyethyl-N'-{[3-methylphenyl]-urea}

N-{1,3-Dihyro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-methylcarboxyethyl-N'-{[3-methylphenyl]-urea},(40 mg) was mixed with 7 mg of lithium hydroxide in 2 ml ofdimethoxyethane and 0.5 ml of water. The reaction mixture was stirredfor 8 hours and concentrated in vacuo. Ethyl acetate was added to theresidue and 1N HCl solution was added until the mixture was neutral. Theethyl acetate extracts were dried (sodium sulfate) and concentrated togive 30 mg of crude product. The crude product was triturated with ethylacetate and petroleum ether to give the title compound: m.p. >175° C.(d).

HPLC=95% pure at 214 nm; TLC R_(f) =0.45 (EtOAc).

NMR (DMSO-D₆): Consistent with structure assignment and confirmspresence of solvent.

FAB MS: 471 (M⁺ +1).

Analysis for C₂₇ H₂₆ N₄ O₄. 0.35 EtOAc.0.15 H₂ O: Calculated: C, 67.67,H, 5.82, N, 11.12. Found: C, 67.70, H, 5.97, N, 11.14.

EXAMPLE 2 Synthesis ofN-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-carboxyethyl-N'-{[3-methylphenyl]-urea}A.1,3-Dihydro-1-methyl-3(R,S)-(methylcarboxyethyl)amino-5-phenyl-1H-1,4-benzodiazepin-2-one

1,3-Dihydro-1-methyl-3(R,S)-amino-5-phenyl-2H-1,4-benzodiazepin-2-one (1g, 377 mole) was dissolved in 10 ml of dry N,N-dimethylformamide andtreated with 1.04 g of solid sodium carbonate at room temperature. Tothis suspension was added 810 mg (3.77 mmole) of methyl 3-iodopropionateand the reaction mixture was stirred overnight. An additional 800 mg ofmethyl 3-iodopropionate and 500 mg of sodium carbonate were added andthe reaction mixture was then heated to 50° C. After 24 hours thereaction mixture was filtered and concentrated under reduced pressure.The residue was partitioned between ethyl acetate and water. The organicphase was washed twice more with water, then dried (sodium sulfate), andconcentrated to yield 720 mg of crude product. The title compound wasobtained as an oil which crystallized on standing after flash silica gelchromatography employing ethyl acetate-hexane (1:1 v/v).Recrystallization from ether afforded the analytical material which hadm.p. 136°-137° C.

B.N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-methylcarboxyethyl-N'-{[3-methylphenyl]-urea}

To a solution of 130 mg (0.37 mmole) of1,3-dihydro-1-methyl-3(R,S)-(methylcarboxyethyl)-amino-5-phenyl-1H-1,4-benzodiazepin-2-onein 2 ml of tetrahydrofuran was added 48 μL of m-toluidine isocyanate atroom temperature. The resulting solution was protected from moisture andstirred for 1 hour. The solvent was removed under reduced pressure andthe residual solid was recrystallized from a methanol-ethylacetate-hexane solvent mixture to give 110 mg of the title compound withm.p. 198° C.

C.N-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-carboxyethyl-N'-{[3-methylphenyl]-urea}

N-[1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-N-methylcarboxyethyl-N'-{[3-methylphenyl]urea}(70 mg) was mixed with 25 mg of lithium hydroxide in 3 ml ofdimethoxyethane and 0.5 ml of water. The reaction mixture was stirredfor 4 hours and concentrated in vacuo. Ethyl acetate and water wereadded to the residue and the resulting mixture was neutralized with 1NHCl solution. The layers were separated and the aqueous layer wasextrated with ethyl acetate. The combined organic extracts were dried(sodium sulfate) and concentrated to give the crude product. The crudeproduct was recrystallized from a methanol-ethyl acetate-hexane solventmixture to give the title compound: m.p. 172°-174° C. (d).

HPLC=99.4% pure at 214 nm; TLC R_(f) =0.48 (EtOAc).

NMR (DMSO-D₆): Consistent with structure assignment and confirmspresence of solvent.

FAB MS: 471 (M⁺ +1).

Analysis for C₂₇ H₂₆ N₄ O₄. 0.40 EtOAc.0.20 H₂ O: Calculated: C, 67.43,H, 5.86, N, 11.00. Found: C, 67.44, H, 5.48, N, 10.98.

What is claimed is:
 1. A compound of Formula I: ##STR7## wherein: R¹ isH, --(CH₂)₂ --CO₂ CH₃ or --(CH₂)₂ --CO₂ H;R² is ##STR8## R³ is absent,one or two of Halogen or CH₃ ; R⁴ is absent, one or two of Halogen orCH₃ ; R⁵ is ##STR9## or the optical isomers, prodrugs orpharmaceutically acceptable salts thereof.
 2. The compound of claim 1,in which the compound isN-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N'-carboxyethyl-N'-{[3-methylphenyl]-ureaorN-{1,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl}-N-carboxyethyl-N'-{[3-methylphenyl]-urea}or a pharmaceutically acceptable salt thereof.